Low Multiplicity Of Infection Research Articles

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

Abstract 2315: Influence of epithelial to mesenchymal transition on viral oncolysis in non small cell lung cancer

Abstract Background: Epithelial to mesenchymal transition (EMT) is distinguished by the loss of epithelial cell junction proteins such as E-cadherin and the relative gain of mesenchymal markers such as vimentin. We hypothesized that EMT may play a role in resistance to viral oncolysis in non-small cell lung cancer (NSCLC). Methods: Multiple human NSCLC cell lines obtained from the American Tissue Culture Collection (H292, H358, H441, H1437, A549, H1703 and SW1573) were evaluated for cell surface expression of E-cadherin via Western blot. Vimentin and Zinc finger E-box binding-1 (Zeb-1) mRNA expression were measured in all of the NSCLC cell lines using RT-PCR to further analyze a molecular profile. Herpes viral infectivity was tested in vitro at varying multiplicity of infection (MOI) using both wild type Herpes (KOS) and an attenuated (ICP-6 defective) HSV-1 mutant (hrR3). Cell surface levels of nectin-1 and herpes virus entry mediator (HVEM) were measured by fluorescence-activating cell sorting (FACS). Results: Western Blots on cell lysates revealed the presence of E-cadherin in H292, H358, H441, and H1437. The cell lines SW1573 and H1703 lacked E-cadherin and expressed higher transcript levels of Vimentin and Zeb-1, suggesting a more mesenchymal phenotype. Cell lines harboring epithelial markers appeared more susceptible to viral oncolysis at low MOI compared to those with mesenchymal markers. The mesenchymal-phenotype cells were 10-1000 fold more resistant to both wild type HSV-1 and replication conditional hrR3, thus suggesting a cell-growth independent mechanism. Viral cytotoxicity did not appear to be Nectin-1 mediated since there was no differential cell surface expression in mesenchymal or epithelial phenotypes. Conclusion: Infectivity and viral oncolysis of NSCLC cell lines may be related to epithelial or mesenchymal phenotype. Nectin-1 does not appear to be the major receptor for viral infectivity in NSCLC. Current and future studies focus on ways to reverse EMT in NSCLC, thus rendering tumor cells more susceptible to viral oncolysis and perhaps chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2315.

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.

Effective Adenovirus-Mediated Gene Transfer Into Neural Stem Cells Derived From Human Embryonic Stem Cells

Human embryonic stem cell-derived neural stem cells (hESC-NSCs) are an attractive cell type for studying aspects of brain development and pathology. To develop the full potential of this model system, it is important to establish a reliable methodology for the manipulation of gene expression in hNSCs. To address this issue, we used an adenoviral vector with a CMV promoter-driven green fluorescent protein (GFP) reporter gene (Ad5-GFP). We optimized conditions for Ad5-GFP infection and assessed the efficiency of infection of whole and dissociated embryonic stem cell (ESC)-derived neurospheres as well as the effect of adenoviral vectors on cell surface marker expression, proliferation, and differentiation potential. Our results demonstrate that most neurosphere cells ( approximately 70%) express the coxsackie and adenovirus receptor and can be infected with Ad5. More specifically, the CD133+ hESC-NSC population could be infected more efficiently than the CD133 population and both populations expressed GFP at high levels. At low multiplicity of infection (MOI < 25), the virus had no significant effect on stem cell marker expression (CD133 and Nestin), cell survival, cell proliferation rate, or differentiation potential. This model system provides a practical new approach to study human NSC function in the context of neurodegenerative and neoplastic disorders.

Combined Chemical-Biological Treatment for Prevention/Rehabilitation of Clogged Wells by an Iron-Oxidizing Bacterium

Groundwater wells containing large concentrations of ferrous iron face serious clogging problems as a result of biotic iron oxidation. Following a short time after their start off, wells get clogged, and their production efficiency drop significantly up to a total obstruction, making cleanup and rehabilitation an economic burden. The present study was undertaken to test an experimental combined treatment (chemical and biological) for future prevention or rehabilitation of clogged wells. Sphaerotilus natans (an iron-oxidizing bacterium) freshly isolated from a deep well was grown to form biofilms on two systems: coupons and sand buried miniature wedge wire screen baskets. A combined chemical-biological treatment, applied at laboratory scale by use of glycolic acid (2%) and isolated bacteriophages against Sphaerotilus natans (SN1 and ER1-a newly isolated phage) at low multiplicity of infection (MOI), showed inhibition of biofilm formation and inactivation of the contaminant bacteria. In addition to complete inactivation of S. natans planktonic bacteria by the respective phages, earlier biofilm treatment with reduced glycolic acid concentration revealed efficient exopolysaccharide (EPS) digestion allowing phages to be increasingly efficient against biofilm matrix bacteria. Utilization of this combined treatment revealed clean surfaces of a model stainless steel wedge wire screen baskets (commonly used in wells) for up to 60 days.

In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.

Improving adeno‐associated vector yield in high density insect cell cultures

Recombinant adeno-associated virus (rAAV) are the most promising vectors for gene therapy. However, large-scale rAAV production remains a challenge for the translation of rAAV-based therapeutic strategies to the clinic. The baculovirus expression vector system (BEVS) has been engineered to produce high rAAV titers in serum-free suspension cultures of insect cells. The typical approach of rAAV production in BEVS has been based on a synchronous infection with three baculoviruses at high multiplicity of infection (MOI) [>3 plaque forming units (pfu)/cell]. An alternative approach is to co-infect at low MOI (0.1 pfu/cell). Both strategies (high and low MOI) were compared at a cell density of 1.0 x 10(6) cells/ml in shake-flask experiments. To increase the rAAV titer, a low MOI combined with an initial cell density at infection of 5.0 x 10(6) cells/ml, in fed-batch mode, was evaluated. Subsequently, the production strategy was validated in 3-l bioreactor runs. An increase of 210% in the rAAV titer (4.7 x 10(11) enhanced transduction units/l) was observed when using low MOI, an effect primarily caused by the increase in cell density. The fed-batch approach resulted in a seven-fold increase of rAAV yield. Controlled operations in bioreactor contributed to further increase the rAAV yield (2.8 x 10(14) vector genomes/l) by 25% in comparison to the shake flask results. This high yield production process using low MOIs and a feeding strategy successfully addresses several limitations of current rAAV production in insect cells and contributes to position the BEVS system as one of the most efficient for large-scale manufacturing of rAAV vectors.

Interferon-γ Regulates the Death ofM. tuberculosis-InfectedMacrophages

We previously described a caspase-independent death induced in macrophages by a high intracellular burden of Mycobacterium tuberculosis (Mtb). This death, with features of apoptosis and necrosis, releases viable bacilli for spreading infection. Interferon (IFN)-γ promotes survival of macrophages with a low intracellular Mtb load by inhibiting bacterial replication. Macrophages in naïve hosts are unable to restrict Mtb replication following aerosol transmission, but IFN-γ is increasingly present when adaptive immunity is expressed in the lungs ~2 weeks post-infection. We therefore investigated the effects of IFN-γ on macrophages challenged with Mtb at high multiplicity of infection (MOI). In contrast to the response at low MOI, IFN-γ accelerated the death of heavily infected macrophages and altered the characteristics of the dying cells. IFN-γ increased caspase-dependent DNA cleavage and apoptotic vesicle formation, but it also increased mitochondrial injury and release of LDH and HMGB1 in a caspase-independent manner. Adaptive immunity in tuberculosis (TB), mediated primarily by IFN-γ, has differential effects on Mtb-induced macrophage cell death depending on the intracellular bacillary load. While IFN-γ generally promotes host defense, our data suggest that its effects on heavily infected macrophages could also accelerate necrosis and spreading infection in TB disease.

Proteasome Inhibitors Decrease AAV2 Capsid derived Peptide Epitope Presentation on MHC Class I Following Transduction

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.

Fiber Mediated Receptor Masking in Non-Infected Bystander Cells Restricts Adenovirus Cell Killing Effect but Promotes Adenovirus Host Co-Existence

The basic concept of conditionally replicating adenoviruses (CRAD) as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI), and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses.

A rapid and efficient method for studies of virus interaction at the host cell surface using enteroviruses and real-time PCR

BackgroundMeasuring virus attachment to host cells is of great importance when trying to identify novel receptors. The presence of a usable receptor is a major determinant of viral host range and cell tropism. Furthermore, identification of appropriate receptors is central for the understanding of viral pathogenesis and gives possibilities to develop antiviral drugs. Attachment is presently measured using radiolabeled and subsequently gradient purified viruses. Traditional methods are expensive and time-consuming and not all viruses are stable during a purification procedure; hence there is room for improvement. Real-time PCR (RT-PCR) has become the standard method to detect and quantify virus infections, including enteroviruses, in clinical samples. For instance, primers directed to the highly conserved 5' untranslated region (5'UTR) of the enterovirus genome enable detection of a wide spectrum of enteroviruses. Here, we evaluate the capacity of the RT-PCR technology to study enterovirus host cell interactions at the cell surface and compare this novel implementation with an established assay using radiolabeled viruses.ResultsBoth purified and crude viral extracts of CVB5 generated comparable results in attachment studies when analyzed with RT-PCR. In addition, receptor binding studies regarding viruses with coxsackie- and adenovirus receptor (CAR) and/or decay accelerating factor (DAF) affinity, further demonstrated the possibility to use RT-PCR to measure virus attachment to host cells. Furthermore, the RT-PCR technology and crude viral extracts was used to study attachment with low multiplicity of infection (0.05 × 10-4TCID50/cell) and low cell numbers (250), which implies the range of potential implementations of the presented technique.ConclusionWe have implemented the well-established RT-PCR technique to measure viral attachment to host cells with high accuracy and reproducibility, at low cost and with less effort than traditional methods. Furthermore, replacing traditional methods with RT-PCR offers the opportunity to use crude virus containing extracts to investigate attachment, which could be considered as a step towards viral attachment studies in a more natural state.

Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions

The Burkholderia cepacia complex (BCC) is made up of at least 17 species of gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3' end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage phiE125 and Burkholderia pseudomallei phage phi1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo.

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKDelta31-47, gKDelta31-68, and gKDelta31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKDelta31-47 and gKDelta31-117 exhibited a gK-null (DeltagK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKDelta31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKDelta31-47 and gKDelta31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBDelta28syn) caused extensive virus-induced cell fusion. However, the gBDelta28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKDelta31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKDelta31-68, DeltagK, or UL20-null virus. The gKa peptide complemented the gKDelta31-47 and gKDelta31-68 mutant viruses for infectious-virus production and for gKDelta31-68/gBDelta28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.

Cleavage Specificity of the UL48 Deubiquitinating Protease Activity of Human Cytomegalovirus and the Growth of an Active-Site Mutant Virus in Cultured Cells

The human cytomegalovirus (HCMV) open reading frame UL48 encodes a 253-kDa tegument protein that is closely associated with the capsid and was recently shown to have ubiquitin-specific protease activity (J. Wang, A. N. Loveland, L. M. Kattenhorn, H. L. Ploegh, and W. Gibson, J. Virol. 80:6003-6012, 2006). Here, we examined the cleavage specificity of this deubiquitinase (DUB) and replication characteristics of an active-site mutant virus. The purified catalytic domain of the UL48 DUB (1 to 359 amino acids), corresponding to the herpes simplex virus UL36(USP) DUB (L. M. Kattenhorn, G. A. Korbel, B. M. Kessler, E. Spooner, and H. L. Ploegh, Mol. Cell 19:547-557, 2005), efficiently released ubiquitin but not ubiquitin-like modifications from a hemagglutinin peptide substrate. Mutating the active-site residues Cys24 or His162 (C24S and H162A, respectively) abolished this activity. The HCMV UL48 and HSV UL36(USP) DUBs cleaved both Lys48- and Lys63-linked ubiquitin dimers and oligomers, showing more activity toward Lys63 linkages. The DUB activity of the full-length UL48 protein immunoprecipitated from virus-infected cells also showed a better cleavage of Lys63-linked ubiquitinated substrates. An HCMV (Towne) mutant virus in which the UL48 DUB activity was destroyed [UL48(C24S)] produced 10-fold less progeny virus and reduced amounts of viral proteins compared to wild-type virus at a low multiplicity of infection. The mutant virus also produced perceptibly less overall deubiquitination than the wild-type virus. Our findings demonstrate that the HCMV UL48 DUB contains both a ubiquitin-specific carboxy-terminal hydrolase activity and an isopeptidase activity that favors ubiquitin Lys63 linkages and that these activities can influence virus replication in cultured cells.

Differential cytokine expression by human dendritic cells in response to different Porphyromonas gingivalis capsular serotypes

Capsular polysaccharides play an important role in the virulence of Gram-positive and Gram-negative bacteria. In Porphyromonas gingivalis, six serotypes have been described based on capsular antigenicity and its pathogenicity has been correlated both in vitro and in animal models. This study aimed to investigate the differential response of human dendritic cells (DCs) when stimulated with different P. gingivalis capsular serotypes. Using different multiplicity of infection (MOI) of the encapsulated strains K1-K6 and the non-encapsulated K(-) strain of P. gingivalis, the mRNA expression levels for interleukin (IL)-1beta, IL-2, IL-5, IL-6, IL-10, IL-12, IL-13, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, and TNF-beta in stimulated DCs were quantified by real-time reverse transcription-polymerase chain reaction. All P. gingivalis capsular serotypes induced a T-helper type 1 (Th1) pattern of cytokine expression. K1- and K2-stimulated DCs expressed higher levels of IL-1beta, IL-6, IL-12p35, IL-12p40, and IFN-gamma and at lower MOI than DCs stimulated with the other strains. These results demonstrate a differential potential of P. gingivalis capsular serotypes to induce DC responses and a higher capacity of strains K1 W83 and K2 HG184 than other K serotypes to trigger cytokine expression.

Oncolytic activity of Sindbis virus in human oral squamous carcinoma cells

Background:Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells.Methods:We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs).Results:Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and IκBα was associated with SIN-induced apoptosis.Conclusion:As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers.

Oncolytic vaccinia therapy of squamous cell carcinoma

BackgroundNovel therapies are necessary to improve outcomes for patients with squamous cell carcinomas (SCC) of the head and neck. Historically, vaccinia virus was administered widely to humans as a vaccine and led to the eradication of smallpox. We examined the therapeutic effects of an attenuated, replication-competent vaccinia virus (GLV-1h68) as an oncolytic agent against a panel of six human head and neck SCC cell lines.ResultsAll six cell lines supported viral transgene expression (β-galactosidase, green fluorescent protein, and luciferase) as early as 6 hours after viral exposure. Efficient transgene expression and viral replication (>150-fold titer increase over 72 hrs) were observed in four of the cell lines. At a multiplicity of infection (MOI) of 1, GLV-1h68 was highly cytotoxic to the four cell lines, resulting in ≥ 90% cytotoxicity over 6 days, and the remaining two cell lines exhibited >45% cytotoxicity. Even at a very low MOI of 0.01, three cell lines still demonstrated >60% cell death over 6 days. A single injection of GLV-1h68 (5 × 106 pfu) intratumorally into MSKQLL2 xenografts in mice exhibited localized intratumoral luciferase activity peaking at days 2–4, with gradual resolution over 10 days and no evidence of spread to normal organs. Treated animals exhibited near-complete tumor regression over a 24-day period without any observed toxicity, while control animals demonstrated rapid tumor progression.ConclusionThese results demonstrate significant oncolytic efficacy by an attenuated vaccinia virus for infecting and lysing head and neck SCC both in vitro and in vivo, and support its continued investigation in future clinical trials.

Regulated and Multiple miRNA and siRNA Delivery Into Primary Cells by a Lentiviral Platform

RNA interference (RNAi) has tremendous potential for investigating gene function and developing new therapies. However, the design and validation of proficient vehicles for stable and safe microRNA (miR) and small interfering RNA (siRNA) delivery into relevant target cells remains an active area of investigation. Here, we developed a lentiviral platform to efficiently coexpress one or more natural/artificial miR together with a gene of interest from constitutive or regulated polymerase-II (Pol-II) promoters. By swapping the stem-loop (sl) sequence of a selected primary transcript (pri-miR) with that of other miR or replacing the stem with an siRNA of choice, we consistently obtained robust expression of the chimeric/artificial miR in several cell types. We validated our platform transducing a panel of engineered cells stably expressing sensitive reporters for miR activity and on a natural target. This approach allowed us to quantitatively assess at steady state the target suppression activity and expression level of each delivered miR and to compare it to those of endogenous miR. Exogenous/artificial miR reached the concentration and activity typical of highly expressed natural miR without perturbing endogenous miR maturation or regulation. Finally, we demonstrate the robust performance of the platform reversing the anergic/suppressive phenotype of human primary regulatory T cells (Treg) by knocking-down their master gene Forkhead Transcription Factor P3 (FOXP3).

Adenovirus vector production using low-multiplicity infection of 293 cells

The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001-0.1 transductional unit (TU) cell(-1). The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell(-1)), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 x 10(5) cells cm(-3) were infected with Ad EGFP at a low MOI of 0.001 TU cell(-1), the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell(-1)) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.